CONSPECTUS Pathogens are recognized by the innate immune system in part via their unique and complex RNA signatures. A key sensor in human innate immunity is the RNA-activated protein kinase PKR, which has two double-stranded RNA (dsRNA) binding motifs (dsRBMs) at its N-terminus. Early studies described PKR as being activated potently by long stretches of perfect dsRNA, a signature typical of viruses. More recently, we and others have found that PKR is also activated by RNAs having structural defects such as bulges and internal loops. This article describes advances in our understanding of the ability of PKR to detect diverse foreign RNAs and how that recognition plays significant roles in discriminating self from non-self. The experiments discussed employ a wide range of techniques including activation assays, native polyacrylamide gel electrophoresis (PAGE), protein footprinting, and small angle X-ray scattering (SAXS). We discuss how misfolding and dimerization of RNA lead to activation of PKR. We also present recent findings on the activation of PKR by varied bacterial functional RNAs including ribozymes and riboswitches, which are among the few structured RNAs known to interact with PKR in a site-specific manner. Molecular models for how these structured RNAs activate PKR are provided. Studies by SAXS revealed that PKR straightens bent RNAs. Most external and internal RNA cellular modifications introduced in vitro and found naturally, such as the m7G cap and m6A group, abrogate activation of PKR, but other modifications, such as 5’-ppp and 2’-fluoro groups, are immunostimulatory and potential anticancer agents. Genome-wide studies of RNA folding in vitro and in vivo have provided fresh insights into general differences in RNA structure amongst bacteria, viruses, and human. These studies suggest that in vivo, cellular human RNAs are less folded than once thought, unwound by helicases, destabilized by m6A modifications, and often bound up with proteins—conditions known to abrogate activation of PKR. It thus appears that non-self RNAs are detected as unmodified, naked RNAs with appreciable secondary and tertiary structure. Observation that PKR is activated by structured but otherwise diverse RNAs is consistent both with the broad-spectrum nature of innate immunity and with the non-specific recognition of RNA by the dsRBM family. These findings provide a possible explanation for the apparent absence of protein-free structured human RNAs, such as ribozymes and riboswitches.
The protein kinase PKR is activated by RNA to phosphorylate eIF-2a, inhibiting translation initiation. Long dsRNA activates PKR via interactions with the dsRNA-binding domain (dsRBD). Weakly structured RNA also activates PKR and does so in a 59-triphosphate (ppp)-dependent fashion, however relatively little is known about this pathway. We used a mutant T7 RNA polymerase to incorporate all four triphosphate-containing nucleotides into the first position of a largely single-stranded RNA and found absence of selectivity, in that all four transcripts activate PKR. Recognition of 59-triphosphate, but not the nucleobase at the 59-most position, makes this RNA-mediated innate immune response sensitive to a broad array of viruses. PKR was neither activated in the presence of g-GTP nor recognized NTPs other than ATP in activation competition and ITC binding assays. This indicates that the binding site for ATP is selective, which contrasts with the site for the 59 end of ppp-ssRNA. Activation experiments reveal that short dsRNAs compete with 59-triphosphate RNAs and heparin for activation, and likewise gel-shift assays reveal that activating 59-triphosphate RNAs and heparin compete with short dsRNAs for binding to PKR's dsRBD. The dsRBD thus plays a critical role in the activation of PKR by ppp-ssRNA and even heparin. At the same time, cross-linking experiments indicate that ppp-ssRNA interacts with PKR outside of the dsRBD as well. Overall, 59-triphosphate-containing, weakly structured RNAs activate PKR via interactions with both the dsRBD and a distinct triphosphate binding site that lacks 59-nucleobase specificity, allowing the innate immune response to provide broad-spectrum protection from pathogens.
The protein kinase PKR is a sensor in innate immunity. PKR autophosphorylates in the presence of dsRNA enabling it to phosphorylate its substrate, eIF2α, halting cellular translation. Classical activators of PKR are long viral dsRNAs, but recently PKR has been found to be activated by bacterial RNA. The features of bacterial RNA that activate PKR are unknown, however. We studied the B. subtilis trp 5’-UTR, which is an indirect riboswitch with secondary and tertiary RNA structures that regulate gene function. Additionally, the trp 5’-UTR binds a protein, TRAP, which recognizes L-tryptophan. We present the first evidence that multiple structural features in this RNA, which are typical of bacterial RNAs, activate PKR in TRAP-free and TRAP/L-Trp-bound forms. Segments from the 5’-UTR, including the terminator, 5’-stem-loop, and Shine-Dalgarno blocking hairpins, demonstrated 5’-triphosphate and flanking RNA tail dependence on PKR activation. Disruption of long-distance tertiary interactions in the 5’-UTR led to partial loss in activation, consistent with highly base-paired regions in bacterial RNA activating PKR. One physiological change a bacterial RNA would face in a human cell is a decrease in the concentration of free magnesium. Upon lowering the magnesium concentration to human physiological conditions of 0.5 mM, the trp 5’-UTR continued to activate PKR potently. Moreover, total RNA from E. coli, depleted of rRNA, also activated PKR under these ionic conditions. This study demonstrates that PKR can signal the presence of bacterial RNAs under physiological ionic conditions and offers a potential explanation for the apparent absence of riboswitches in the human genome.
The protein kinase, PKR, is activated by long stretches of double-stranded (ds) RNA. Viruses often make long dsRNA elements with imperfections that still activate PKR. However, due to the complexity of the RNA structure, prediction of whether a given RNA is an activator of PKR is difficult. Herein, we systematically investigated how various RNA secondary structure defects contained within model dsRNA affect PKR activation. We find that bulges increasingly disfavor activation as they are moved toward the center of a duplex and as they are increased in size. Model RNAs designed to conform to cis, trans, or bent global geometries through strategic positioning of one or more bulges decreased activation of PKR relative to perfect dsRNA, although cis-bulged RNAs activated PKR much more potently than trans-bulged RNAs. Activation studies on bulge-containing chimeric duplexes support a model wherein PKR monomers interact adjacently, rather than through-space, for activation on bulged substrates. Last, unusually low ionic strength induced substantial increases in PKR activation in the presence of bulged RNAs suggesting that discrimination against bulges is higher under biological ionic strength conditions. Overall, this study provides a set of rules for understanding how secondary structural defects affect PKR activity.
The innate immune system provides the first line of defense against pathogens through the recognition of non-specific patterns in RNA to protect the cell in a generalized way. The human RNA-activated protein kinase, PKR, is a dsRNA binding protein and an essential sensor in the innate immune response, which recognizes viral and bacterial pathogens through their RNAs. Upon activation via RNA-dependent autophosphorylation, PKR phosphorylates the eukaryotic initiation factor eIF2α leading to termination of translation. PKR has a well-characterized role in recognizing viral RNA, where it binds long stretches of double-stranded RNA non-sequence specifically to promote activation; however, the mechanism by which bacterial RNA activates PKR and the mode by which self RNA avoids activating PKR are unknown. We characterized activation of PKR by three functional bacterial RNAs with pseudoknots and extensive tertiary structure: the cyclic-di GMP riboswitch, the glmS riboswitch-ribozyme, and the twister ribozyme, two of which are ligand-activated. These RNAs were found to activate PKR with comparable potency to long dsRNA. Enzymatic structure mapping in the absence and presence of PKR reveals a clear PKR footprint and provides a structural basis for how these bacterial RNAs activate PKR. In the case of the cyclic di-GMP riboswitch and the glmS riboswitch-ribozyme, PKR appears to dimerize on the peripheral double-stranded regions of the native RNA tertiary structure. Overall, these results provide new insights into how PKR acts as an innate immune signaling protein for the presence of bacteria and suggest a reason for the apparent absence of protein-free riboswitches and ribozymes in the human genome.
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